Novel 3-(oxygenated alkyl)-1,9-dihydroxy and 1-hydroxy-9-keto dibenzo[b,d]py

ABSTRACT

1-Hydroxy-9-keto-3-alkyl-dibenzo[b,d]pyrans or 1,9-dihydroxy-3-alkyl-dibenzo[b,d]pyrans are oxygenated on the penultimate carbon of the alkyl side chain by fermentation with a strain of the micro-organism Bacillus cereus.

BACKGROUND OF THE INVENTION

U.S. Pat. Nos. 3,822,188, 3,808,234, and 3,864,492 all to Fager et al.disclose the fermentation of Δ⁹ -THC(1-hydroxy-3-n-pentyl-6,6,9-trimethyl-6a,7,10,10a-tetrahydrodibenzo[b,d]pyran)with such micro-organisms as Cunninghamella blakesleeana, Streptomycesviridoilavus, Mucor parasiticus, Aspergillus fonsecaeus, etc. to producethe corresponding 4'-hydroxy derivatives; i.e., compounds in which then-pentyl side-chain is oxidized on its penultimate carbon by the actionof the micro-organism. The compounds thus produced are said to beanti-depressants. Vidic et al., U.S. Pat. No. 3,897,306, oxidize anisomer (Δ⁸ -THC) with Streptomyces lavendulae or with Peliculiariafilamentosa to produce a quite different compound in which the C-7carbon is hydroxylated to yield a 7-hydroxy-Δ⁸ -THC derivative.Robertson, et al., Biomedical Mass Spectrometry, 1975(2) 266-271 findthat exposure of Δ⁹ -THC, Δ⁸ -THC, cannabinol, and cannabidiol to thefungus Syncephalastrum racemasum yields products which carry a hydroxylgroup on the penultimate carbon atom.

A series of1-hydroxy-3-alkyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[,d]pyran-9-oneshave been found by Archer to be useful as anti-depressant, anti-anxiety,sedative, and analgesic drugs--see U.S. Pat. No. 3,928,598. Compounds ofthis structure were first synthesized by Fahrenholtz, et al., J. Am.Chem. Soc., 88 2078 (1966), 89, 5934 (1967), as intermediates for thepreparation of Δ⁸ - or Δ⁹ -THC. (See also U.S. Pat. No. 3,507,885 andU.S. Pat. No. 3,636,058). The same group of Δ⁸ - and Δ⁹ -THC compoundsare disclosed by Petrzilka in U.S. Pat. No. 3,873,576.

SUMMARY OF THE INVENTION

This invention provides a novel group of side-chain oxygenateddibenzo[b,d]pyrans of the following formula: ##STR1## wherein X and Yare either both hydrogen or one is hydrogen and the other methyl;wherein, when taken singly, one of the pair R and R' and one of the pairR" and R'" is H and the other is OH and, when taken together, the pair Rand R' and the pair R" and R'" form the oxygen of a ketone group; andwherein n is 1, 2, 3 or 4.

The above compounds are prepared by subjecting a compound of thefollowing formula: ##STR2## wherein R, R', X, Y and n have the samemeaning as hereinabove, to the oxygenating activity of a micro-organismof the species Bacillus cereus. The oxygenating action of the Bacilluscereus species upon the dibenzopyran-1-ol-9-ones or dibenzopyran-1,9-diols substrates can take place under standard submergedculture fermentation conditions, or the growing micro-organism cells canbe harvested, resuspended, and the substrate added to the resultingsuspension.

The novel products according to Formula I above produced during thefermentation were isolated by standard extraction procedures and werepurified by high performance liquid chromatography and/or preparativethin-layer chromatography. The structures were determined by NMRspectroscopy, mass spectroscopy, UV spectroscopy and infra-redspectroscopy. Compounds preparable by the above procedure include thefollowing:

Fromtrans-1-hydroxy-3-(1',2'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-9-one.

a.trans-1,6',9-trihydroxy-3-(1',2'-dimethyl-heptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo-[b,d]pyran.

b.trans-1,9-dihydroxy-3-(1',2'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]-pyran-6'-one.

c.trans-1,6'-dihydroxy-3-(1',2'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]-pyran-9-one.

d.(±)-trans-1-hydroxy-3-(1',2'-dimethylheptyl)-6,6-dimethyl)-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]-pyran-6',9-dione.

Fromtrans-1-hydroxy-3-(1',1'-dimethyloctyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]-pyran-9-one.

a.trans-1,7',9-trihydroxy-3-(1',1'-dimethyl-octyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran.

b.trans-1,9-dihydroxy-3-(1',1'-dimethyloctyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-7'-one.

c.trans-1,7'-dihydroxy-3-(1',1'-dimethyloctyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-9-one.

d.(±)-trans-1-hydroxy-3-(1',1'-dimethyloctyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-7',9-dione.

From cis-1-hydroxy-3-(1',1'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-9-one.

a.cis-1,6',9-trihydroxy-3-(1',1'-dimethylheptyl)-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran.

b.cis-1,6'-dihydroxy-3-(1',1'-dimethylheptyl)-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-9-one.

c.cis-1,9-dihydroxy-3-(1',1'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-6'-one.

d. (±)cis-1-hydroxy-3-(1',1'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-6',9-dione.

Fromtrans-1-hydroxy-3-(1',1'-dimethylpentyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-9-one.

a.trans-1,4',9-trihydroxy-3-(1',1'-dimethyl-pentyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo-[b,d]pyran.

b.trans-1,9-dihydroxy-3-(1',1'-dimethylpentyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]-pyran-4'-one.

c.trans-1,4'-dihydroxy-3-(1',1'-dimethylpentyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]-pyran-9-one.

d. (±)-trans-1-hydroxy-3-(1', 1'-dimethylpentyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo [b,d]pyran-4',9-dione.

Other compounds coming within the scope of Formula I above include:

cis-1,5'-dihydroxy-1', 1', 2',6,6-pentamethyl-3-hexyl -6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-9-one.

cis-1',2', 6,6-tetramethyl-3-hexyl-6,6a, 7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-1,5',9-triol.

cis-1',2',6,6-tetramethyl-3-pentyl-6,6a, 7,8,10,10a-hexahydro-9H-dibenzo [b,d]pyran-1,4', 9-triol.

cis-1',2',6,6-tetramethyl-3-pentyl-6,6a, 7,8,10,10a-hexahydro-9H-dibenzo [b,d]pyran-1,4'-diol-9-one and the like.

Compounds according to Formulas I or II above contain asymmetric centersat 6a, and 10a and at 9 when one of R or R' is hydroxyl and the other ishydrogen. In addition, there may be asymmetric centers in the side-chainalkyl group as, for example, when R' is 1,2-dimethylheptyl, twoasymmetric centers are present in the side-chain at C₁ ' and C₂ '. TheFahrenholtz synthetic procedure described above in which the double bondisomerizes from the Δ^(6a)(10a) position to the Δ¹⁰(10a) positionproduces a racemate in which C_(6a) is asymmetric, the hydrogen beingeither above or below the plane of the dibenzopyran fused-ring system.Hydrogenation of the Δ¹⁰(10a) double bond with, for example, an activemetal in liquid ammonia produces a second asymmetric center at C_(10a),but the hydrogen which adds to this carbon under the hydrogenation orreduction conditions will usually take the more favorable transconfiguration relative to the hydrogen at C_(6a) with a lesser quantityof compound of the cis configuration being produced. Reduction of theketone group at C₉ yields a mixture of isomers in which the hydroxylgroup is in the axial (9α) or equatorial (9β) configuration. Thus acompound in which the side chain contains no asymmetric centers, as forexample 1,9-dihydroxy-3-(1',1'-dimethylheptyl) -6,6-dimethyl-6,6a,7,8,1010a-hexahydro-6H-dibenzo[b,d]pyran, will occur as four racemates orracemic pairs to give a total of 8 stereoisomers. Compounds such as1,9-dihydroxy-3-(1',2'-dimethylheptyl)-6,6-dimethyl-6,6a,7,,10,10a-hexahydro-6H-dibenzo[b,d]pyran containing two additionalasymmetric centers in the side chain will have a total of fiveasymmetric centers, those at 6a,9,10a and at C₁ ' and C₂ ' in the sidechain, yielding altoghether 32 possible isomers occuring as 16racemates.

Compounds having a ketone group at C₉ will, of course, have one lessasymmetric center. For example,1-hydroxy-3-(1',1'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-6H-dibenzo[b,d]pyran-9-one would occur as 4stereoisomers instead of 8 with the 9-hydroxy analogs.

In addition, the process of this invention whereby the penultimatecarbon atom of the alkyl side chain is hydroxylated produces anadditional asymmetric carbon. This hydroxylation is believed to bestereoselective; i.e., one of two possible isomers is predominately, ifnot virtually entirely, produced.

If the end product of the process of this invention is a side-chainketone, there would be no additional asymmetric carbon and the number ofstereoisomers unchanged from the number present in the substrate. Thefermentation process of this invention can also produce an asymmetriccenter at C₉ by reduction of a C₉ ketone employed as a substrate therebyincreasing by one the number of asymmetric carbons. Again, thisreduction is believed to be predominately stereospecific in that onlyone of the C₉ hydroxy isomers is produced; namely the 9S hydroxy isomer.

The compounds of this invention according to Formula I above areprepared as follows:

A lyophilized culture of Bacillus cereus, NRRL B-8172 is grown in avegetative medium containing glucose, minerals, and yeast extract plusother ingredients. The culture is then transferred to a larger flaskcontaining the same growth medium as before, and the organism is againincubated for a period of from one to two days. The chosen substrate canbe added to the culture at this point or a cell suspension can beprepared by harvesting the cells from the growth medium and resuspendingthem in sterile buffer. The substrate is usually added as an ethanolsolution and the fermentation carried on for a period of from 4 to 8days. Any conversion products plus residual starting material arerecovered by extracting the culture medium before or after filtration ofthe cells with a water immiscible solvent such as ethyl acetate. Theorganic extracts thus obtained are combined, washed with buffer, dried,filtered and then concentrated to dryness in vacuo. The products of thefermentation isolated in this residue are then separated by columnchromatography.

Alternatively, the oxidation-reduction enzyme systems present in thecells can be isolated by lysing the cells, removing cell debris byfiltration and collecting the isolated enzymes in the filtrate. Asbefore, the substrate is added to the conversion system, in this case anisolated enzyme system, in proper concentrations and the reactionmixture shaken until a substantial amount of products according toFormula I above are formed.

This invention is more fully illustrated by the following specificexample.

EXAMPLE

A soil isolate designated as Bacillus cereus NRRL B-8172 (A36659) wasgrown for 48-96 hours on a slant culture at a temperature in the rangeof 25°-30° C. using the following medium: Beef extract 1.5 g.; peptone6.0 g.; yeast extract, 3.0 g.; agar, 20 g.; deionized water, 1 liter.The medium was autoclaved for 15 minutes at 121° C. (15 psi) prior touse. The culture was maintained on these slants at 4° C. The cells from48-96 hour old slant culture were then lyophilized and stored at 4° C.The lyophilized cells were used to inoculate a flask of the followingmedium:

Glucose -- 30 g.

Yeast extract -- 1.0 g.

(NH₄)₂ SO₄ -- 10 g.

Na₂ SO₄ -- 0.5g.

K₂ hpo₄ -- 5.0 g.

MgSO₄.7 H₂ O -- 0.4 g.

FeSO₄.7 H₂ O -- 0.02 g.

MnSO₄.4 H₂ O -- 0.02 g.

NaCl -- 0.02 g.

H₃ bo₃ -- 0.5 mg.

CuSO₄.5 H₂ O -- 0.04 mg.

Na₂ MoO₄.2 H₂ O -- 0.2 mg.

ZnSO₄.7 H₂ O -- 8.0 mg.

CaCl₂ -- 0.05 mg.

CoCl₂.6 H₂ 0 -- 0.2 mg.

Deionized H₂ O -- 1.0 l.

The pH of the medium was adjusted to 7.0 with concentrated aqueoushydrochloric acid, and was dispensed in 50 or 100 ml. portions into 250or 500 ml. erlenmeyer flasks respectively and each flask stoppered witha cotton plug. Each erlenmeyer flask containing medium was autoclaved asbefore. The autoclaved flasks were then inoculated. After inoculationwith a lyophilized culture of NRRL B-8172 the culture flask wasincubated at 30° C. on a rotary shaker (250 rpm 2.5 inch stroke) for24-48 hours.

Cells were harvested from the above fermentations by centrifugation at2300 rpm for 20-30 minutes at 4° C. The supernatant was separated bydecantation, and the packed cells were washed with 0.1 M phosphatebuffer at pH 6-7. The washed cells were then resuspended in a smallvolume buffer at pH=7.0, or a similar buffer containing 3 percentgulcose. The resulting suspension was divided into 100 ml. to 125 ml.portions in sterile 500 ml. erlenmeyer flasks. 80 mg. ofdl-trans-1-hydroxy-3-(1',1'-dimethylheptyl)-6',6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-9-onedissolved in 1.5 ml. of ethanol were added to each flask of cellsuspension. After addition, the culture was further incubated for from4-8 days. The incubated flasks were combined, and the combined cells andmedium extracted four times, each time with one-half volume of ethylacetate. The ethyl acetate extracts were combined, washed twice withone-tenth volume of water, dried, and the ethyl acetate therefromremoved by evaporation in vacuo. The resulting residue containedunmodified starting ketone plus a mixture of five fermentation-inducedoxidation products of the starting ketone. The residue was subjected tohigh pressure liquid chromatography using a Merck silica gel 60 prepackcolumn, equilibrated in chloroform under 2-6 psi of nitrogen. Theresidue was disolved in a small volume of chloroform and injected intothis column which was chromatographed batch wise with solvent mixturescontaining increasing concentrations of ethyl acetate. Column fractionsshown to contain the same conversion product of the ketone substrate onTLC were pooled. The conversion product contained therein was furtherpurified by preparative thin-layer chromatography as follows; theresidual material obtained by evaporation of the pooled fractions todryness was dissolved in ethyl acetate. The ethyl acetate solution wasspotted on a silica gel thin-layer plate using 15-20 mg. sample perplate. The plates were developed in a 1:1 benzene:ethyl acetate solventsystem, air-dried, and observed under ultra-violet light to mark theareas of the conversion product. These areas were eluted with chloroformand ethyl acetate to yield purified products.

The following are further typical runs made in accordance with the aboveprocedure.

RUN I

Substrate:dl-trans-1-hydroxy-3-(1',1'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-9-one

Amount: 160 mg.

Incubation Time: 5 days

Separation: -- Chromatography over Silica gel

Conversion:

    ______________________________________                                                        Eluting                                                       Products                                                                              R.sub.f Solvent           Weight                                      ______________________________________                                        A       .500    CHC1.sub.3         5.5 mg.                                    C       .232    CHC1.sub.3, 2% EtOAc-                                                                           10.1 mg.                                                    98% CHC1.sub.3                                                D        179    10% EtOAc--90% CHC1.sub.3                                                                       17.1 mg.                                    ______________________________________                                    

Substrate anddl-trans-1,9-dihydroxy-3-(1',1'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-(6aR,10aR,9Risomer-reduction product of 9-keto group to an α-hydroxyl) were alsoisolated. Compounds A, C and D were isolated as yellow oils.

RUN II

Substrate: -- Same as I

Amount: -- 320 mg.

Incubation Time: -- 5 days

Separation: -- Same as I

Conversion:

Products

A, C, D -- See I

B,R_(f) -- 0.348, eluted with CHCl₃, 17.5 mg.

RUN III

Substrate: -- Same as I

Amount: -- 800 mg.

Incubation Time: -- 5 days

Separation: -- Same as I

Conversion:

Products

A, B, C, D

Also a quantity of 1,9-dihydroxy derivative, reduction product of thesubstrate.

COMPOUND A(±)-trans-3-(1',1'-Dimethylheptyl)-6,6a,7,8,10,10a-hexahydro-1-hydroxy-6,6-dimethyl-9H-dibenzo[b,d]pyran-6',9-dione

Uv(etOH) λ_(max) 208, 230 (shoulder) and 280 nm (ε=38000, 10000, and200); ir (CHCl₃) 3.04 (OH) and 5.88μ (C═O); ¹ H NMR (CDCl₃) δ6.77 (s,1H, exchanges with D₂ O), 6.34, 6.30 (2d, 1H each, J=2Hz, H₂ and H₄),4.08 (broad d, 1H, J=14Hz, H₁₀α), 3.03-1.02 (30H) especially 2.35 (t,2H, J=7Hz, H₅ '), 2.07 (s, 3H, CH₃ C═O), 1.47 (s, 3H, 6β-CH₃), 1.20 (s,6H gem di-CH₃ 's), and 1.11 ppm (s, 3H, 6α-CH₃); an exact massdetermination gave m/e 386.2461 (calcd for C₂₄ H₃₄ O₄, 386.2457).

COMPOUND B(+)-trans-3-(1',1'-Dimethylheptyl)-6,6a,7,8,10,10a-hexahydro-1,6'-dihydroxy-6,6-dimethyl-9H-dibenzo[b,d]pyran-9-one

UV(EtOH) λ_(max) 207, 225 (shoulder), and 280 nm (ε=16800, 4200, and80); ir (CHCl₃) 3.03 (OH) and 5.90μ (C═O); ¹ H NMR (CDCl₃) δ 6.34 (s,2H, H₂ and H₄), 4.08 (broad d, 1H, J=14Hz, H₁₀α), 3.74 (m, 1H, H₆ '),3.02-1.02 (32H) especially 1.47 (s, 3H, 6β--CH₃), 1.20 (s, 6H, gemdi-CH₃ 's), 1.15 (d, 3H, J=6Hz, CH₃ C-OH), and 1.11 ppm (s, 3H,6α--CH₃); [α]²⁵ D + 46.3° (OC₃, CHCl₃); and exact mass determinationgave m/e 388.2614 (calcd for C₂₄ H₃₆ O₄, 388.2613).

COMPOUND Ctrans-3-(1',1'-Dimethylheptyl)-6,6a,7,8,10,10a-hexahydro-1,9-dihydroxy-6,6-dimethyl-9H-dibenzo[b,d]pyran-6'-one

UV(EtOH) λ_(max) 208, 227 (shoulder), and 280 nm (ε=19000, 4600, and80); ir (CHCl₃) 2.98 (OH) and 5.88μ (C═O); ¹ H NMR (CDCl₃) δ 6.34, 6.28(2d, 1H each, J=2Hz, H₂ and H₄); 4.29 (broad s, 1H, H₉ equatorial), 3.27(broad d, 1H, J=15Hz, H₁₀ equatorial), 3.0-1.02 (32H) especially 2.96(broad s, 1H, H_(10a)), 2.37 (t, 3H, CH₂ --C═O), 2.08 (s, 3H, CH₃ C═O),1.39 (s, 3H, 6β--CH₃), 1.19 (s, 6H, gem di-CH₃ 's), and 1.06 ppm (s, 3H,6α--CH₃); an exact mass determination gave m/e 388.2614 (calcd for C₂₄H₃₆ O₄, 388.2613 ).

COMPOUND Dtrans-3-(1',1'-Dimethylheptyl)-6,6a,7,8,10,10a-hexahydro1,6',9-trihydroxy-6,6-dimethyl-9H-dibenzo[b,d]pyran

UV(EtOH) λ_(max) 208, 225 (shoulder), and 275 nm (ε=32000, 7300, and130); ir (CHCl₃) 2.98μ (OH); ¹ H NMR (CDCl₃) 6.34, 6.31 (2d, 1H each,J=2Hz, H₂ and H₄), 4.28 (broad s, 1H, H₉ equatorial), 3.74 (m, 1H, H₆'), 3.25 (broad d, 1H, J=15Hz, H₁₀ equatorial), 3.02-1.02 (33H)especially 2.96 (broad s, 1H, H_(10a)), 1.37 (s, 3H, 6β-CH₃), 1.19 (s,6H, gem di-CH₃ 's), 1.15 (d, 3H, J=6Hz, CH₃ CH-OH), and 1.06 ppm (s, 3H,6α--CH₃); and exact mass determination gave m/e 390.2769 (calcd for C₂₄H₃₈ O₄, 390.2770).

CHARACTERIZATION OF BACILLUS CEREUS NRRL B8172

This bacterium is classified as a strain of Bacillus cereus Franklandand Frankland. It is a large, spore-forming rod, averaging 4.7 μ × 1.6 μand occurs in short chains. It is gram positive to gram variable anddoes nort appear to be motile. Endospores are ellipsolidal and occurprincipally central to paracentral. Spores are produced readily within18-24 hours on nutrient agar. Spores are 0.95 μ × 1.7 μ. Sporangia arenot swollen.

Colonies are slightly roughened, irregular, whitish-opaque, withundulate margins. The colony surface is dull, lacking a distinctivesheen.

The optimum temperature for growth is 30°. Growth occurs between 8° and37° C. No growth occurs at 43° C.

In ammonium salts medium acid but no gas is produced from D-glucose andD-maltose. Neither acid nor gas is produced with L-arabinose,D-mannitol, and D-xylose. It does not produce either indole or H₂ S.Gelatin is liquified rapidly within 24 hours, starch is hydrolyzed,peptonization of milk and acid production is slow (6 days). The cultureis catalase positive and produces acetyl methyl carbinol.

This culture differs from Bacillus megaterium by producing acetyl methylcarbinol and by not producing acid from mannitol.

As previously stated, substitution of other substrates according toFormula II above for(±)-trans-1-hydroxy-3-(1',1'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-9-onesuch as(±)-trans-3-(1',1'-dimethylheptyl)-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-1,9diol,in the above fermentation yields the analogous four products: productshaving either a ketone or hydroxyl on the penultimate carbon of the sidechain and either a ketone or hydroxyl at position 9 of thedibenzo[b,d]pyran ring. Similarily other organisms can be employed inplace of Bacillus cereus NRRL B-8172 including such organisms asCumminghamella blakeselleeana, Cunninghamella elegans, Streptomycescinnamonous, Mucor parasiticus, etc.

The compounds of this invention are useful as analgesics. Theiranalgesic activity is shown by their ability to inhibit writhing in miceinduced by the intraperitoneal injection of acetic acid. Inhibition ofanalgesic writhing is a standard laboratory test for analgesic activity.Estimated ED₅₀ (dose sufficient to inhibit 50 percent of the writhings)for the above compounds are as follows: Compound A, greater than 20mg/kg; Compound B, 8.0 mg/kg; Compound C, 4.2 mg/kg; and Compound D, 2.0mg/kg.

In addition to their utility as analgesics, some of the compounds ofthis invention are useful as intermediates for producing other compoundsof the invention by reduction or oxidation. For example, compound A, acompound containing ketone functions at position 9 of thedibenzo[b,d]pyran ring system and on the penultimate carbon can bereduced by sodium borohydride to yield compound D which containshydroxyls on the same two carbons. Similarly, compounds C and B can bereduced to yield compound D.

In employing the compounds of this invention as analgesics, the activedrug can be mixed with a pharmaceutically-acceptable diluent and themixture filled into empty telescoping gelatin capsules so that eachcapsule contains one analgesic does of the particular drug. A personrequiring analgesia can then take from one to four of these capsules oneto four times a day as prescribed by the physician. Other pharmaceuticalformulations such as tablets, suspensions, and the like can also beemployed.

We claim:
 1. The fermentation process which comprises subjecting as asubstrate a compound of the formula ##STR3## wherein X and Y are eitherboth hydrogen or one is hydrogen and the other methyl, wherein n is 1,2, 3 or 4 and wherein, when taken singly, one of R and R' is H and theother is OH and when taken together, form the oxygen of a ketone groupto the oxygenating activity of a strain of the micro-organism Bacilluscereus to produce an oxygenated compound of the formula ##STR4## whereinX and Y are either both hydrogen or one is hydrogen and the othermethyl;wherein, when taken singly, one of the pair R and R' and one ofthe pair R" and R'" is H and the other is OH and, when taken together,the pair R and R' and the pair R" and R'" form the oxygen of a ketonegroup, the pairs R, R' and R", R'" being the same or different; andwherein n is 1, 2, 3, or
 4. 2. A process according to claim 1 whereinthe oxygenating micro-organism is Bacillus cereus NRRL B-8172.
 3. Aprocess according to claim 1 wherein the substrate is(+)-trans-1-hydroxy-3-(1',1'-dimethylheptyl)-6,6-dimethyl-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-9-one.4. A process according to claim 1 wherein the substrate is(±)-trans-3-(1',1'-dimethylheptyl)-6,6a,7,8,10,10a-hexahydro-9H-dibenzo[b,d]pyran-1,9-diol.